IMPROVED SENSOR MEMBRANES

Description

Field of the Invention

[0001] The present invention relates to electrode membrane combinations for use in ion selective electrodes and biosensors. In addition, the present invention relates to methods for the production of such electrode membrane combinations and the use of ion selective electrodes and biosensors incorporating such electrode membrane combinations in the detection of analytes. The present invention also relates to novel compounds used in the electrode membrane combinations.

Background of the Invention

[0002] Lipid bilayer membranes (also known as black lipid membranes - BLM's) are well known in the biological and chemical fields. The ability of ionophores to modulate the ion flux through these membranes is also well known. Modulation of the ion flux of the membrane in response to specific molecules is also known, especially in the biochemical fields. The lipid bilayer membranes are however extremely fragile and sensitive to non-specific physical and chemical interference. The preparation and properties of the BLM's are fully described in textbooks and literature articles.

[0003] It has been known since 1967 that ionophores incorporate into lipid bilayers (P. Mueller et al, Biochem. Biophys. Res Commun., 26 (1967) 298; A.A. Lev et al Tsitologiya, 9 (1967) 102;) in BLM's and that the selective ion flux through the membrane could thus be monitored. Possibility of producing a lipid bilayer containing ionophores on an ionic hydrogel reservoir and using such as an ion selective electrode has also been suggested (U.J. Krull et al, US Patent No 4,661,235, Apr. 28, 1987), however no means of obtaining reproducible and stable bilayer membranes have been taught in the art. Using a Langmuir-Blogett bilayer and multilayer approach (T.L. Fare et al Powder Technology, 3, (1991), 51-62; A. Gilardoni et al, Colloids and Surfaces, 68, (1992), 235-242) has been attempted however the ion selectivity was inadequate and the response time was too slow for practical purposes, stability was not adequate and the LB technique is generally considered to be too difficult for industrial applications.

[0004] Ionophores in the context of the present invention are any of the naturally occurring lipophilic bilayer membrane compatible ion carriers such as valinomycin, nonactin, methyl monensin or other naturally occurring ion carriers, or synthetic ionophores such as lipophilic coronands, cryptands or podands, or low molecular weight (<5000 g/mol) naturally occurring or synthetic ion channels such as gramicidin, alamethicin, mellitin or their derivatives. Additionally trialkylated amines or carboxylic acids such as phytic acid may serve as proton ionophores.

[0005] Ion channels may also include large, lipid membrane compatible, protein ion channels, especially where their function and stability is enhanced through their incorporation into lipid bilayers that are essentially free of extraneous alkane material.

[0006] In the broad context of the present invention lipids are deemed to be any amphiphilic molecules, either naturally occurring or synthetic, containing a hydrophobic hydrocarbon group and a hydrophilic head group.

[0007] Biosensors and ion selective electrodes incorporating gated ionophores in lipid membrane combinations have been disclosed in Patent Application Nos EP-A-0 382 736, EP-A-0 432 188, EP-A-0 455 705 and WO-A-92 17 788.

[0008] As is disclosed in these applications, suitably modified receptor molecules may be caused to co-disperse with amphiphilic molecules and produce membranes with altered surface binding propertie which are useful in the production of biosensor receptor surfaces of high binding ability and high binding specificities. It is also disclosed that ionophores such as polypeptide ionophores may be co-dispersed with amphiphilic molecules, thereby forming membranes with altered properties in relation to the permeability of ions. There is also disclosure of various methods of gating these ion channels such that in response to the binding of an analyte the conductivity of the membrane is altered. The applications also disclose methods of producing membranes with improved stability and ion flux using chemisorbed arrays of amphiphilic molecules attached to an electrode surface and means of producing lipid membranes incorporating ionophores on said chemisorbed amphiphilic molecules. Additionally, means of co-dispersing ion selective ionophores with amphiphilic molecules thereby producing ion selective membrane combinations are disclosed.

[0009] The present inventors have now determined improved means of increasing the stability and ion flux properties of the lipid membranes through the use of novel synthetic lipids and lipid combinations, and novel means of membrane assembly.

[0010] In various embodiments the present invention consists in the use of novel bilayer membrane spanning lipids and bilayer lipids and methods of assembly thereof, in order to modulate the properties of the lipid sensor membrane so as to control the ion transport properties of the ionophore, the thickness and fluidity of the membrane, the stability of the membrane, the response to serum, plasma or blood, and the non-specific absorption of proteins to the membrane. In various embodiments the present invention consists in novel synthetic lipids including linker lipids for use in attaching a membrane including a plurality of ionophores to an electrode and providing a space between the membrane and the electrode in which the membrane is either in part or totally made up of the linker lipid, the linker lipid comprising within the same molecule a hydrophobic region capable of spanning the membrane, an attachment group used to attach the molecule to an electrode surface, a hydrophilic region intermediate said hydrophobic region and the attachment group, and a polar head group region attached to the hydrophobic region at a site remote from the hydrophilic region.

[0011] Preferably the head group region is selected from the group consisting of groups normally associated with naturally occurring or synthetic lipids such as glycerol, phosphatidyl choline, phosphatidyl ethanolamine, mono-, di- or tri-methylated phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, disubstituted head groups as found in cardiolipins, ganglioside head groups, sphingomyelin head groups, plasmalogen head groups, glycosyl, galactosyl, digalactosyl, sulfosugar, phosphosugar, N-acetyl neuramic acid, sialic acid, aminosugar head groups, carbohydrate head groups, gal(betal-3)galNAc(betal-4) [NAcNeu(alpha2-3]gal(betal-4)glc-ceramide, oligomers of ethylene glycol, ethylene glycol, oligomers of propylene glycol, propylene glycol, amino acids, oligomers of amino acids, combinations of oligomers of ethylene glycol or propylene glyco functionalised with amino acids or other ionic species or any combination or derivative of the above.

[0012] It is generally preferred that the head group is a naturally occurring or synthetic head group that can be used to minimise the non-specific binding of proteins onto the surface of the membrane.

[0013] In order to provide surface characteristics that minimise the non-specific binding of proteins, it is preferred that the head group is a polyethylene glycol ranging in molecular weight of between 600-6000 g/mol.

[0014] In a further preferred embodiment, the head group is a phosphatidyl choline group.

[0015] In a further preferred embodiment, the head group is a glycerol head group.

[0016] In a further preferred embodiment, the head group is a biotin or a biotinylated 6-aminocaproic acid group or an N-biotinylated oligomer of 6-aminocaproic acid.

[0017] In a further preferred embodiment, the head group is a Gal(betal-3)galNAc(betal-4)[NAcNeu(alpha2-3]gal(betal-4-Glc-ceramide head group.

[0018] In a further preferred embodiment, it is preferred that the head group is a group capable of being used to covalently link a protein molecule onto the linker lipid. The protein molecule may be either a receptor such as an antibody or an antibody fragment or may be an enzyme or may be a protein molecule chosen in order to impart biocompatible properties to the membrane.

[0019] It is further preferred that the head group is terminated in a carboxylic acid group capable of being used to conjugate the linker lipid with a protein molecule via the amine groups on the protein.

[0020] It is further preferred that the head group is a polyethylene glycol in the molecular weight range 400-1000g/mol terminated in a carboxylic acid group.

[0021] In a further preferred embodiment, the head group is a group capable of being covalently linked with a protein molecule via the aldehyde groups generated from the oxidation of carbohydrate groups on the protein molecule.

[0022] In a further preferred embodiment, the head group includes a hydrazide derivative.

[0023] In a further preferred embodiment, the head group is a polyethylene glycol terminated in a carboxy hydrazide derivative.

[0024] In a further preferred embodiment, the head group is a group capable of being covalently linked to a protein molecule via free thiol groups on the protein molecule.

[0025] It is further preferred that the head group includes a maleimide derivative.

[0026] In a further preferred embodiment, the head group is a group capable of being covalently coupled to a carboxylic acid group on a protein molecule.

[0027] The hydrophobic group has the general structure as shown in Figure 1 where the group (X) is a hydrocarbon chain that is approximately half the length of the group (Y) or are absent and wherein the group (Y) in Figure 1 is a single chain hydrocarbon group of length between 20-60 Å long and contains a rigid spacer group.

[0028] It is preferred that the group (X) will generally be between 10-22 carbons in length and may be a saturated, unsaturated or polyunsaturated hydrocarbon, or may be an alkyl substituted hydrocarbon such as the phytanyl group or other mono- or permethylated hydrocarbon chain.

[0029] It is further preferred that the group (X) is a phytanyl group.

[0030] In a further preferred embodiment the group (Y) contains within the chain a rigid spacer group such as biphenyl ether or biphenylamine or other biphenyl compound. The rigid spacer group serves the function of making the synthesis of the group simpler as it easily enables the coupling of two smaller alkyl chains onto the rigid spacer group, enabling long sections of the group (Y) to be synthesised readily. The rigid spacer group also enhances the ability of the linker lipid to assume the membrane spanning conformation of the linker lipid as opposed to an U-shaped conformation within the membrane.

[0031] In a further preferred embodiment, the group (Y) is a single chain group that is between 30-50 Å long and contains within the chain a N,N'-alkyl substituted 4,4'-biphenyl amine group.

[0032] In a further preferred embodiment, the group (Y) is a single chain group that is between 30-50 Å long and contains within the chain a 4,4'-biphenyl ether group.

[0033] In a further preferred embodiment, the group (Y) is a bis-hexadecyl 4,4'-biphenyl ether.

[0034] In a further preferred embodiment, the group (Y) is a bis-tetradecyl 4,4'-biphenyl ether.

[0035] In a further preferred embodiment, the group (Y) is a bis-dodecyl 4,4'-biphenyl ether.

[0036] In a further preferred embodiment, the group (Y) is a single chain group that is between 20-60 Å long and contains within the chain an alkyl substituted amine.

[0037] In a further preferred embodiment, the group (Y) consist in a single chain group that is between 20-60 Å long and contains within the chain a bis-alkylated pentaerythritol group.

[0038] In a further preferred embodiment of the present invention, the membrane spanning lipid is a single chain lipid in which the group (X) in Figure 1 is absent.

[0039] In a further preferred embodiment, the group (Y) contains groups that can alter their conformation in response to an external stimulus such as light, pH, redox chemistry or electric field. The change in conformation within the group (Y) will allow the properties of the membrane such as thickness to be controlled through such external stimulus. This can in turn be used to modulate the conduction of ion channels through modulation of the on/off times of the channels and the diffusion of the channels.

[0040] In a preferred embodiment, where the group (Y) alters its conformation in response to light stimulus, the group (Y) contains a 4,4'- or 3,3'-disubstituted azobenzene.

[0041] In a further preferred embodiment the group (Y) contains a group that undergoes a spiropyran-merocyanine equilibrium in response to light stimulus.

[0042] In a further preferred embodiment of the present invention, the hydrophobic region of the linker lipid consists of oligomers of long chain amino acids, such as 11-aminoundecanoic acid, 16-aminohexadecanoic acid or other amino acid where the carbon chain is preferably between 6-20 carbons long, and where the amino acids are linked via amide linkages.

[0043] It is further preferred that the amide groups are tertiary, alkyl substituted amide groups, where the alkyl groups are phytanyl groups or saturated or unsaturated alkyl groups between 1-18 carbons in length.

[0044] The nature of the hydrophilic group, the attachment group and the electrode are as described in WO-A-9217788.

[0045] As is set out in this earlier application it is preferred that the attachment region of the linker lipid is attached to the electrode surface by chemisorption. In a situation where the electrode is formed of a transition metal such as gold, platinum, palladium or silver, it is preferred that the attachment region includes thiol, disulphide, sulphide, thione, xanthate, phosphine or isonitrile groups.

[0046] In further preferred embodiment the electrode is formed of gold, silver, platinum or palladium and the attachment region includes either a thiol or a disulfide group, the linker lipid being attached to the electrode by chemisorption.

[0047] In an alternate embodiment where the electrode is formed such that a hydroxylated surface is formed on the electrode, it is preferred that the attachment region includes silyl groups such as silyl-alkoxy or silyl chloride groups. The hydroxylated electrode surface may be a prepared by a number of techniques known to someone skilled in the art and may consist of oxidised silicon or oxidised metals such as tin, platinum, iridium.

[0048] In yet a further preferred embodiment the electrode is formed of oxidized silicon, tin, platinum or iridium and the attachment region includes silyl groups, the linker lipid being attached to the electrode by covalent attachment.

[0049] The hydrophilic region of the linker lipid is preferably a long chain hydrophilic compound. The hydrophilic region of the linker lipid may be composed of oligo/poly ethers, oligo/poly peptides, oligo/poly amides, oligo/poly amines, oligo/poly esters, oligo/poly saccharides, polyols, multiple charged groups (positive and/or negative), electroactive species or combinations thereof. The main requirement of the hydrophilic region of the linker lipid is that it allows the diffusion of ions through the ionophores provided in the membrane. This is achieved by the placement of suitable ion and/or water binding sites along or within the length of the long chain that makes up the reservoir region.

[0050] In a preferred embodiment of the invention the hydrophilic region consists of an oligoethylene oxide group. The oligoethylene oxide group may consist of four to twenty ethylene oxide units.

[0051] In a further preferred embodiment the hydrophilic region consists of a subunit of tetraethylene glycol attached to succinic acid. This tetraethylene glycol/succinic acid subunit may be repeated 1-4 times.

[0052] In a further preferred embodiment the hydrophobic region of a proportion of the linker lipids have covalently attached thereto an ionophore via a hydrophobic spacer.

[0053] As set out above the molecule having a hydrophobic region as shown in Fig. 1 incorporating a rigid spacer group provides a number of advantages. This hydrophobic region can, of course, be synthesized separately from the hydrophilic region, attachment region, and polar head group region. This hydrophobic region, or synthetic lipid, is believed to be new in its own right and can be included in bilayer membranes as a membrane spanning lipid to improve various characteristics of the membrane, such as stability.

[0054] Accordingly, in a first aspect the present invention consists in a synthetic lipid for use in bilayer membranes, the synthetic lipid comprising a structure as shown in Figure 1 in which Y is a single chain group that is between 20 and 60Å long and contains a rigid spacer group and X are hydrocarbon chains approximately half the length of Y or are absent.

[0055] It is preferred that the group (X) will generally be between 10-22 carbons in length and may be a saturated, unsaturated or polyunsaturated hydrocarbon, or may be an alkyl substituted hydrocarbon such as the phytanyl group or other mono- or permethylated hydrocarbon chain.

[0056] It is further preferred that the group (X) is a phytanyl group.

[0057] In a further preferred embodiment the rigid spacer group is a biphenyl ether or biphenylamine or other biphenyl compound.

[0058] In a further preferred embodiment, the group (Y) is a single chain group that is between 30-50 Å long and contains within the chain a N,N'-alkyl substituted 4,4'-biphenyl amine group.

[0059] In a further preferred embodiment, the group (Y) is a single chain group that is between 30-50 Å long and contains within the chain a 4,4'-biphenyl ether group.

[0060] In a further preferred embodiment, the group (Y) is a bis-hexadecyl 4,4'-biphenyl ether.

[0061] In a further preferred embodiment, the group (Y) is a bis-tetradecyl 4,4'-biphenyl ether.

[0062] In a further preferred embodiment, the group (Y) is a bis-dodecyl 4,4'-biphenyl ether.

[0063] In a further preferred embodiment, the group (Y) is a single chain group that is between 20-60 Å long and contains within the chain an alkyl substituted amine.

[0064] In a further preferred embodiment, the group (Y) consist in a single chain group that is between 20-60 Å long and contains within the chain a bis-alkylated pentaerythritol group.

[0065] In a further preferred embodiment of the present invention X in Figure 1 is absent.

[0066] In a further preferred embodiment the synthetic lipid includes a head group. Preferred head groups are those listed previously in the disussion of linker lipids.

Detailed Description of the Invention

[0067] In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following examples.

Example 1

Synthesis of membrane spanning lipids

[0068] 1,3-Benzylidine glycerol was prepared according to the method of H.S. Hill et al in Carbohydrates and Polysaccharides, 50, (1928), 2242-2244. The 1,3-benzylidine glycerol was then treated with sodium hydride in tetrahydrofuran and phytanyl bromide under reflux for 24 hours to give the glycerol 2-phytanylether 1,3-benzylidine. This ether was then treated with a mixture of potassium borohydride and boron trifluoride etherate in refluxing tetrahydrofuran for 24 hours to give the glycerol 1-benzylether 2-phytanylether. The product was then treated with sodium hydride and 1,16-dibromohexadecane in refluxing tetrahydrofuran for 24 hours to yield the glycerol 1-benzylether 2-phytanylether 3-(16-bromohexadecyl) ether. The homologous compounds using 1,12-dibromododecane, or 1,14-dibromotetradecane were produced in similar fashion. Treatment of the product with biphenol and sodium hydride in refluxing tetrahydrofuran gave the bis-benzyl protected membrane spanning lipid which, after isolation, was debenzylation using palladium on charcoal to give the diol shown in Figure 2. Addition of a reservoir component as described in PCT/AU92/00132 in the presence of dicyclohexylcarbodiimide and dimethylamino pyridine gave the membrane spanning lipid shown in Figure 3 which contains an alcohol head group (MSL-OH). Treatment of this membrane spanning lipid with the diacidchloride of an acid functionalised polyethylene glycol 400 (average molecular weight 400g/mol) followed by an aqueous workup gave the membrane spanning lipid shown in Figure 4 (MSLPEG400COOH) which contains a dicarboxy polyethylene glycol 400 head group. Treatment of the diol shown in Figure 2 with firstly, one equivalent of BOC-glycine/dicyclohexylcarbodiimide and dimethylamino pyridine and isolation of the monosubstituted compound, secondly with trifluoroacetic acid to remove the BOC group, thirdly treatment with a biotin-xx-N-hydroxysuccinimide (where the X group is an 6-aminocaproic acid group), and fourthly, treatment of the product with the reservoir component as above in the presence of dicyclohexylcarbodiimide and dimethylamino pyridine gave the membrane spanning lipid as shown in Figure 5 (MSLXXB).

Example 2

Method of adsorbing Gramicidin B

1st layer

[0069] Onto freshly prepared 2mm² gold electrodes was deposited 2µl of a ethanolic solution containing 140mM glycerol monoleate, 140µM reservoir lipid A, 14µM MSLXXB, 1.4µM Gramicidin B. 100µl 0.1M NaCl was immediately added and the assembly allowed to stand overnight. The saline solution was then removed, the assembly rinsed with ethanol (5 x 100µl) and drained.

2nd layer

[0070] To the above prepared electrode was added 5µl of a ethanolic solution of 140mM glycerol monooleate and 1.4µ M biotin-gramicidin conjugate, 2%(v/v) tetradecane. The assembly was immediately treated with 100µl 0.1M NaCl. The saline solution is removed and replaced with fresh saline (100µl) five times.

[0071] Figure 6 shows the impedance of the electrodes before (a), and after (b) challenge with 1µl 0.05mg/ml streptavidin solution (0.1M NaCl). The impedance trace obtained for the sealed membrane, i.e. without gramicidin derivative in the 2nd layer, is shown in (c).

[0072] Conducting membranes that respond to the addition of streptavidin can also be obtained by varying the method described above with the following:

1) type of membrane spanning lipid added

2) replacing glycerol monooleate with other different chain length derivatives or glycerol monooleate ether derivatives

3) the concentration of MSLXXB from 1µM to 140mM

4) the concentration of Gramicidin B from 1µM to 14µM

5) replacing reservoir phytanyl lipid B with reservoir lipid A or reservoir phytanyl lipid C-in the concentration range 10µM to 1mM.

6) saline can be omitted from the first layer, and glycerol monooleate can also be omitted from the first layer

7) ethanol can be replaced with other polar solvents such as methanol or dioxane

8) the 2nd layer can be made up with or without addition of alkanes such as tetradecane.

[0073] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as claimed. The present examples are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. A synthetic lipid for use in bilayer membranes, the synthetic lipid having the general structure

in which Y is a single chain group that is between 20 and 60Å long and contains a rigid spacer group and X are hydrocarbon chains approximately half the length of Y or are absent

2. A synthetic lipid as claimed in claim 1 in which the group (X) will be between 10-22 carbons in length and may be a saturated, unsaturated or polyunsaturated hydrocarbon, or may be an alkyl substituted hydrocarbon such as a phytanyl group or other mono- or permethylated hydrocarbon chain.

3. A synthetic lipid as claimed in claims 1 or 2 in which the group (X) is a phytanyl group.

4. A synthetic lipid as claimed in claims 1, 2 or 3 in which the rigid spacer group is biphenyl ether, biphenylamine or other biphenyl compound.

5. A synthetic lipid as claimed in any one of claims 1 to 4 in which the group (Y) is a single chain group that is between 30-50 Å long and contains within the chain a N,N'-alkyl substituted 4,4'-biphenyl amine group or 4,4'-biphenyl ether group.

6. A synthetic lipid as claimed in any one of claims 1 to 5 in which the group (Y) is bis-hexadecyl 4,4'-biphenyl ether, bis-tetradecyl 4,4'-biphenyl ether or bis-dodecyl 4,4'-biphenyl ether.

7. A synthetic lipid as claimed in any one of claims 1 to 6 in which the group (Y) is a single chain group that is between 20-60 Å long and contains within the chain an alkyl substituted amine or bis-alkylated pentaerythritol group.

8. A synthetic lipid as claimed in any one of claims 1 or 4 to 7 in which X is absent.

9. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group region is a polyethylene glycol ranging in molecular weight of between 600-6000g/mol.

10. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group is a phosphatidyl choline group, a glycerol head group, biotin, a biotinylated 6-aminocaproic acid group, N-biotinylated oligomer of 6-aminocaproic acid, a Gal(betal-3)galNAc(betal-4)[NAcNeu(alpha2-3]gal(betal-4)-Glc-ceramide head group, a polyethylene glycol terminated in a carboxy hydrazide derivative, or a group capable of being covalently linked to a protein molecule via free thiol groups on said protein molecule.

11. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group is terminated in a carboxylic acid group capable of being used to conjugate the synthetic lipid with a protein molecule via the amine groups on the protein.

12. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group is a polyethylene glycol in the molecular weight range 400-1000g/mol terminated in a carboxylic acid group.

13. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group is a group capable of being covalently linked with a protein molecule via aldehyde groups generated from the oxidation of carbohydrate groups on the protein molecule.

14. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group includes a hydrazide derivative.

15. A synthetic lipid as claimed in any one of claims 1 to 8 in which the head group includes a maleimide derivative.

16. A synthetic lipid as claimed in any one of claims 1 to 15 in which the group (Y) contains groups that can alter their conformation in response to an external stimulus such as light, pH, redox chemistry or electric field.

17. A membrane including the synthetic lipid as claimed in any one of claims 1 to 16.

Ansprüche

1. Synthetisches Lipid zur Verwendung in Zweischichtmembranen, wobei das synthetische Lipid die allgemeine Struktur

aufweist, in der Y eine einkettige Gruppe ist, die 20 bis 60 Å lang ist und eine starre Abstandsgruppe enthält, und X Kohlenwasserstoffketten sind, die ungefähr die Hälfte der Länge von Y aufweisen oder nicht vorhanden sind.

2. Synthetisches Lipid nach Anspruch 1, bei dem die Gruppe (X) 10 bis 22 Kohlenstoffatome in der Länge aufweist und ein gesättigter, ungesättigter oder mehrfach ungesättigter Kohlenwasserstoff sein kann oder ein alkylsubstituierter Kohlenwasserstoff, wie beispielsweise eine Phytanylgruppe oder eine andere mono- oder permethylierte Kohlenwasserstoffkette sein kann.

3. Synthetisches Lipid nach Anspruch 1 oder 2, bei dem die Gruppe (X) eine Phytanylgruppe ist.

4. Synthetisches Lipid nach Anspruch 1, 2 oder 3, bei dem die starre Abstandsgruppe Biphenylether, Biphenylamin oder eine andere Biphenylverbindung ist.

5. Synthetisches Lipid nach einem der Ansprüche 1 bis 4, bei dem die Gruppe (Y) eine Einkettengruppe ist, die 30 bis 50 Å lang ist und innerhalb der Kette eine N,N'-alkylsubstituierte 4,4'-Biphenylamingruppe oder 4,4'-Biphenylethergruppe enthält.

6. Synthetisches Lipid nach einem der Ansprüche 1 bis 5, bei dem die Gruppe (Y) Bishexadecyl-4,4'-biphenylether, Bis-tetradecyl-4,4'-biphenylether oder Bisdodecyl-4,4'-biphenylether ist.

7. Synthetisches Lipid nach einem der Ansprüche 1 bis 6, bei dem die Gruppe (Y) eine Einkettengruppe ist, die 20 bis 60 Å lang ist und innerhalb der Kette eine alkylsubstituierte amin- oder bisalkylierte Pentaerythritgruppe enthält.

8. Synthetisches Lipid nach einem der Ansprüche 1 oder 4 bis 7, bei dem X nicht vorhanden ist.

9. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppenregion ein Polyethylenglykol mit einem Molekulargewichtsbereich von 600 bis 6000 g/mol ist.

10. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppe ein Phosphatidylcholingruppe, eine Glycerin-Kopfgruppe, Biotin, eine biotinylierte 6-Aminocapronsäuregruppe, ein N-biotinyliertes Oligomer von 6-Aminocapronsäure, eine Gal(betal-3)galNAc(betal-4)[NAcNeu(alpha2-3]gal(betal-4)-Glc-ceramid-Kopfgruppe, ein Polyethylenglykol, das in einem Carboxyhydrazinderivat terminiert ist, oder eine Kopfgruppe ist, die in der Lage ist, kovalent mit einem Proteinmolekül über freie Thiolgruppen an dem Proteinmolekül verbunden zu sein.

11. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppe in einer Carbonsäuregruppe terminiert ist, die in der Lage ist, zum Konjugieren des synthetischen Lipids mit einem Proteinmolekül über die Aminogruppen des Proteins verwendet zu werden.

12. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppe ein Polyethylenglykol im Molekulargewichtsbereich von 400 bis 1000 g/mol ist und in einer Carbonsäuregruppe terminiert ist.

13. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppe eine Gruppe ist, die in der Lage ist, kovalent mit einem Proteinmolekül über Aldehydgruppen verbunden zu werden, die durch Oxidation von Kohlenhydratgruppen an dem Proteinmolekül erzeugt werden.

14. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppe ein Hydrazidderivat umfasst.

15. Synthetisches Lipid nach einem der Ansprüche 1 bis 8, bei dem die Kopfgruppe ein Maleimidderivat umfasst.

16. Synthetisches Lipid nach einem der Ansprüche 1 bis 15, bei dem die Gruppe (Y) Gruppen enthält, die ihre Konformation in Reaktion auf einen externen Stimulus wie Licht, pH-Wert, Redox-Chemie oder elektrisches Feld verändern können.

17. Membran, die das synthetische Lipid gemäß einem der Ansprüche 1 bis 16 enthält.

Revendications

1. Lipide synthétique à utiliser dans des membranes bicouches, le lipide synthétique ayant la structure générale :

   dans laquelle Y est un groupe à chaîne unique qui a une longueur comprise entre 20 et 60 Å et contient un groupe écarteur rigide et X sont des chaînes hydrocarbonées faisant approximativement la moitié de la longueur de Y, ou sont absents.

2. Lipide synthétique selon la revendication 1, dans lequel le groupe (X) va avoir une longueur comprise entre 10 et 22 atomes de carbone et peut être un hydrocarbure saturé, insaturé ou polyinsaturé ou peut être un hydrocarbure à substitution alkyle tel qu'un groupe phytanyle ou une autre chaîne hydrocarbonée mono- ou perméthylée.

3. Lipide synthétique selon la revendication 1 ou 2, dans lequel le groupe (X) est un groupe phytanyle.

4. Lipide synthétique selon la revendication 1, 2 ou 3, dans lequel le groupe écarteur rigide est le biphényléther, la biphénylamine ou un autre composé biphényle.

5. Lipide synthétique selon l'une quelconque des revendications 1 à 4, dans lequel le groupe (Y) est un groupe à chaîne unique qui a une longueur comprise entre 30 et 50 Å et contient à l'intérieur de la chaîne un groupe 4,4'-biphényléther ou un groupe 4,4'-biphénylamine à substitution N,N'-alkyle.

6. Lipide synthétique selon l'une quelconque des revendications 1 à 5, dans lequel le groupe (Y) est le bis-hexadécyl-4,4'-biphényléther, le bis-tétradécyl-4,4'-biphényléther ou le bis-dodécyl-4,4'-biphényléther.

7. Lipide synthétique selon l'une quelconque des revendications 1 à 6, dans lequel le groupe (Y) est un groupe à chaîne unique qui a une longueur comprise entre 20 et 60 Å et contient à l'intérieur de la chaîne un groupe pentaérythritol bis-alkylé ou amine à substitution alkyle.

8. Lipide synthétique selon l'une quelconque des revendications 1 à 4 et 7, dans lequel X est absent.

9. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel la région de groupe de tête est un polyéthylèneglycol ayant une masse moléculaire située dans la plage comprise entre 600 et 6000 g/mol.

10. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel le groupe de tête est un groupe phosphatidylcholine, un groupe de tête glycérol, la biotine, un groupe acide 6-aminocaproïque biotinylé, un oligomère N-biotinylé d'acide 6-aminocaproïque, un groupe de tête Gal (β1-3) galNAc (β1-4) [NAcNeu (α2-3)]gal [β1-4)-Glc-céramide, un polyéthylèneglycol se terminant en un dérivé carboxyhydrazide, ou un groupe capable d'être lié de façon covalente à une molécule de protéine par l'intermédiaire de groupes thiol libres sur ladite molécule de protéine.

11. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel le groupe de tête se termine en un groupe acide carboxylique capable d'être utilisé pour conjuguer le lipide synthétique avec une molécule de protéine par l'intermédiaire des groupes amine sur la protéine.

12. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel le groupe de tête est un polyéthylèneglycol ayant une masse moléculaire située dans la plage comprise entre 400 et 1000 g/mol et se terminant en un groupe acide carboxylique.

13. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel le groupe de tête est un groupe capable d'être lié de façon covalente avec une molécule de protéine par l'intermédiaire de groupes aldéhyde générés par l'oxydation de groupes hydrate de carbone sur la molécule de protéine.

14. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel le groupe de tête comprend un dérivé d'hydrazide.

15. Lipide synthétique selon l'une quelconque des revendications 1 à 8, dans lequel le groupe de tête comprend un dérivé de maléimide.

16. Lipide synthétique selon l'une quelconque des revendications 1 à 15, dans lequel le groupe (Y) contient des groupes qui peuvent altérer leur conformation en réponse à un stimulus externe tel que la lumière, le pH, une chimie redox ou un champ électrique.

17. Membrane comprenant le lipide synthétique selon l'une quelconque des revendications 1 à 16.

Drawing